Share this article on:

Carrageenan/MIV-150 (PC-815), a Combination Microbicide

Fernández-Romero, José A. BS*; Thorn, Mitchell MS*; Turville, Stuart G. PhD*; Titchen, Kanani BS*; Sudol, Kristin BS*; Li, Jifan PhD*; Miller, Todd BS*; Robbiani, Melissa PhD*; Maguire, Robin A. BS*; Buckheit, Robert W. Jr PhD†; Hartman, Tracy L. MS†; Phillips, David M. PhD*

Sexually Transmitted Diseases: January 2007 - Volume 34 - Issue 1 - pp 9-14
doi: 10.1097/01.olq.0000223287.46097.4b

Objective: The objective of this article is to study the effect of PC-815, a novel combination microbicide containing carrageenan and the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150, in blocking HIV-1 and HIV-2 infections in vitro as compared with Carraguard alone.

Goal: The goal of this study was to develop a combination microbicide that is more efficacious than Carraguard against HIV-1 and HIV-2.

Study Design: The microtiter syncytial assay was used to evaluate: 1) the antiviral and virucidal activity of MIV-150 against HIV-1MN; 2) the additive effect of MIV-150 when combined with carrageenan; and 3) a possible interference of seminal fluid in the antiviral activity of these compounds.

Results: MIV-150 effectively inactivated free virus. Combination of MIV-150 and Carraguard demonstrated an additive antiviral effect. Seminal fluid had no effect on the antiviral activity of MIV-150 or Carraguard. The average concentration that blocks 50% of infection (EC50) for PC-815 was approximately 10 times stronger than Carraguard for the different clinical isolates used in the study.

Conclusion: Theoretically, PC-815 is likely to be a more efficacious microbicide than Carraguard.

PC-815, a new combination microbicide containing carrageenan and the nonnucleoside reverse transcriptase inhibitor MIV-150, demonstrates virucidal activity and is more efficacious in blocking HIV-1 and HIV-2 in vitro than Carraguard or MIV-150 alone.

From the *Population Council, New York, NY; and †ImQuest BioSciences Inc., Frederick, Maryland

The authors thank Bo Oberg and Disa Bottinger for sharing their knowledge of MIV-150, and Carrie Van Driel for her work on the manuscript.

The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: CEM-SS from Dr. Peter L. Nara; TZMbl cells from Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme, Inc.; human rIL-2 from Dr. Maurice Gately, Hoffmann-LaRoche Inc.; HIV-1MN and clinical isolates 98/CN/009, 93/MW/959, 93/MN/960, 98/TZ/013, 98/TZ/017, 97/ZA/003, 97/ZA/009, 97/ZA/012, 93/MW/965, and 98/CN/006 from WHO-UNAIDS; and HIV-2CDC310342 from Dr. Mark Rayfield for Dr. Stephen Wiktor.

Supported by NIH grant PO1-HD41752 and U.S. Agency for International Development.

Correspondence: David M. Phillips, PhD, Population Council, 1230 York Avenue, New York, NY 10021. E-mail:

Received for publication October 18, 2005, and accepted March 17, 2006.

OUR LABORATORY HAS PREVIOUSLY DEVELOPED the topical microbicide, Carraguard, which contains the sulfated polysaccharide carrageenan. The inherent nature of carrageenan is such that it gives Carraguard the rheologic properties desirable for a vaginal microbicide1 and is itself an active antiviral and antibiotic ingredient. In vitro, carrageenan has been shown to block HIV-1.2 In animal systems, Carraguard has been shown to prevent: 1) rhesus macaques from vaginal infection by SHIV (R. Black, personal communication, and Stuart G. Turville, Todd Miller, and Melissa Robbiani, unpublished observations); 2) cell trafficking across the vaginal epithelium3; 3) vaginal infection by herpes simplex virus type 24; 4) vaginal colonization by Neisseria gonorrhoeae4; and 5) human papillomavirus from transforming human xenographs (M.K. Howett, personal communication). In addition, carrageenan has a strong safety profile5–7 and is remarkably stable.6,8,9 In 5 phase 1 and 2 expanded safety studies in humans, Carraguard was safe and acceptable when applied vaginally.10

In addition to carrageenan, several other sulfated polymers have been shown to be active in blocking HIV infection of target cells by T-tropic viruses in vitro.11–13 Given that these sulfated polymers are negatively charged molecules, it is believed that their mechanism of action is to bind to the positively charged region of the viral envelope.14–16 Because the envelope of CCR5 viruses (M-tropic) is less positively charged than CXCR4 viruses (T-tropic),17,18 there is controversy over whether sulfated polymers will be effective in blocking CCR5 viruses. Ability to block CCR5 viruses is critical for a microbicide, because even if both phenotypes are transmitted together, it is the CCR5 viruses that are amplified during the initial infection.19 In addition, the majority of microbicide trials are being planned to take place in sub-Saharan Africa where the majority of infections occur through CCR5 viruses with a high prevalence of clade C viruses.20

MIV-150 is a nonnucleoside reverse transcriptase inhibitor (NNRTI) developed by Medivir AB (Huddinge, Sweden) for use as an antiviral therapeutic. The mechanism of action of NNRTIs is as an allosteric inhibitor of HIV reverse transcriptase (RT), which blocks viral DNA elongation thereby blocking viral replication. In vitro studies demonstrate 3 important characteristics of MIV-150. First, MIV-150 is a tight-binding HIV-RT enzyme inhibitor characterized by a rapid formation and slow dissociation rate and is effective at inactivating clinical isolates of HIV at very low concentrations. Second, Medivir scientists have demonstrated that MIV-150 inactivates viruses that are resistant to other antiviral drugs, including NNRTIs, nucleoside reverse transcriptase inhibitors (NRTIs), and protease inhibitors. Finally, resistance to MIV-150 develops much more slowly than resistance to several other currently marketed NNRTIs (Disa Bottinger, Bo Oberg, personal communication on MIV-150).

Because NNRTIs inhibit HIV strains independent of the virus phenotype, MIV-150 is likely to inhibit both T-tropic and M-tropic viruses. In support of this, studies were performed using clinical isolates from patients who have never been treated with NNRTIs and that represent HIV-1 clades A, B, C, and E. The activity of MIV-150 was similar against these clinical isolates from the 4 different subtypes of HIV. Additional studies carried out in the rhesus macaque model indicates that systemic injection of MIV-150 can prevent the onset of viremia after vaginal challenge with an M-tropic RT-SHIV (Disa Bottinger, Bo Oberg, personal communication).

Extensive toxicology studies carried out in mice, rats, dogs, and monkeys indicated that MIV-150 had no identified toxicity in the dose range studied. Human studies indicated that, although MIV-150 is well tolerated, it had poor systemic absorption after oral administration and, thus, is not suitable for antiviral therapy. Although the property of poor systemic absorption is a disadvantage for an oral therapeutic, it is an advantage for a microbicide that needs to be present and active in the vagina. Subsequently, under a contractual agreement, the Population Council was given exclusive rights for use of MIV-150 in a microbicide.

A series of experiments were conducted to confirm the active concentration of MIV-150 with those values established by Medivir to evaluate if MIV-150 can inactivate free virus and to determine if the combination of MIV-150 and Carraguard (PC-815) is more effective against both M-tropic and T-tropic HIV-1 than Carraguard. Further experiments compared Carraguard and PC-815 for activity against HIV-2 and activity in the presence of seminal fluid.

Back to Top | Article Outline

Materials and Methods

Preparation of PC-815

A 100 μmol/L stock solution of MIV-150 (Chiron Corporation, Emeryville, CA) was prepared using 100% ethanol. PC-815, which contains 10 μmol/L of MIV-150 and 3% carrageenan (PDR98-15; FMC BioPolymer, Philadelphia PA) in phosphate buffer was prepared by mixing desired concentrations of MIV-150 stock solution and carrageenan solution. Methylcellulose 2.5% in phosphate buffer was used as a negative control for antiviral activity testing.

Back to Top | Article Outline

Antiviral Activity of MIV-150

To evaluate the antiviral activity of MIV-150, the microtiter syncytial assay as described by Nara21 was used to determine the ability of MIV-150 to: 1) protect targets cell from becoming infected and 2) inactivate free virus. The assay uses CEM-SS cells, which were obtained from NIH AIDS Research and Reference Reagent Program (Rockville, MD). Cells were maintained in RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS), 2 μmol/L L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco-BRL, Bethesda, MD).

Back to Top | Article Outline

Protection of Target Cells From Infection.

The HIV-1MN (NIH AIDS Research and Reference Reagent Program) virus inoculum (between 100 and 200 syncytial forming units [SFU]) and different concentrations of MIV-150 were simultaneously added to CEM-SS cells. After 3 to 4 days of infection, the number of SFU was quantified.

Back to Top | Article Outline

Inactivation of Free Virus.

Two hundred syncytial forming units of HIV-1MN were incubated with various concentrations of MIV-150 in a final volume of 200 μL of RPMI culture medium in a 96-well flat-bottomed plate (Falcon; BD Biosciences, San Jose, CA) for 1 hour at 37°C, 5% CO2, and 98% humidity. Centriprep YM-30 filtration columns (Millipore, Billerica, MA) were used to remove unbound MIV-150 from the viral inoculum as described by Motakis and Parniak.22 Briefly, 200 μL of viral inoculum and MIV-150 were diluted in 15 mL RPMI and centrifuged for 20 minutes at 1500 g at 4°C. The filtration step was repeated, and the viral inoculum was concentrated to approximately 500 μL. A volume of 150 μL of the viral inoculum was added to 50 μL of CEM-SS cells to perform the microtiter syncytial assay (4 × 104 cells/well).

Back to Top | Article Outline


The colorimetric assay based on the reduction of a tetrazolium salt (2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide [XTT])23 was used to determine the cellular cytotoxicity of Carraguard and PC-815.

Back to Top | Article Outline

Additive Effect of MIV-150

Various concentrations of MIV-150 were mixed with different concentrations of carrageenan. The assay was used to identify any additive effect in the anti-HIV activity when MIV-150 is combined with carrageenan. The ability of the compounds to block HIV-1MN infection of TZM-bl cells (NIH AIDS Research and Reference Reagent Program) was obtained using the detection of β-galactoside activity in infected cells as described by Derdeyn et al.24

Back to Top | Article Outline

Activity Against Clinical Isolates of HIV

Primary clinical isolates (98/CN/009, 93/MW/959, 93/MW/960, 98/TZ/013, 98/TZ/017, 97/ZA/003, 97/ZA/009, 97/ZA/012, 93/MW/965, and 98/CN/006) of clade C HIV-1 (all of which are R5 viruses; however, 93/MW/959 and 93/MW/960 do not induce syncytia) were obtained from the NIH AIDS Research and Reference Reagent Program and were titered and evaluated in PBMC infection assay by monitoring the RT activity in the supernatant of infected cells as previously described.25 Carraguard and PC-815 were evaluated in this assay.

Back to Top | Article Outline


Strain HIV-2CDC310342 (NIH AIDS Research and Reference Reagent Program) is a primary isolate from an asymptomatic blood donor from the Cote D’Ivoire. The virus stock was 10−4/0.025 mL with an RT concentration of 10 ng/mL as determined by Lenti RT Activity Assay (Cavidi Tech, Uppsala, Sweden). Microbicidal activity of the different compounds was tested by using 100 TCID50 of virus in the presence of different concentrations of the compounds and activated PBMCs. The plate was then centrifuged at 2910 rpm for 99 minutes at 23°C and the wells washed 5 times with RPMI. Growth medium with 20 U/mL IL-2 was added next (NIH AIDS Research and Reference Reagent Program), and the plate was incubated at 37°C in 5% CO2 and 98% humidity. After 3 days, the RT activity in the supernatant was measured using a Lenti RT Activity Assay (Cavidi Tech) and the percentage about the virus control calculated. The experiment was repeated 3 times. The EC50 was calculated, and the median and standard deviation for the 3 experiments for MIV-150, Carraguard, and PC-815 was obtained.

Back to Top | Article Outline

Effect of Seminal Fluid on Anti-HIV Activity

Seminal fluid (SF) was obtained by centrifugation of semen, from healthy donors, to remove spermatozoa.26 Aliquots of SF were stored at −20°C until needed. To evaluate the full impact that SF may have on the activity of Carraguard and MIV-150, separate mixtures of undiluted SF were mixed with Carraguard, MIV-150, or phosphate-buffered saline (PBS; control). Additional controls were Carraguard or MIV-150 in PBS. Carraguard and MIV-150 were each diluted in SF or PBS at a concentration of 4 mg/mL and 400 μmol/L, respectively. They were then incubated for 30 minutes at 37°C. After incubation, mixtures were diluted to concentrations such that MIV-150 and Carraguard were still effective against HIV, but the toxicity and anti-HIV activity of SF were no longer present. The CEM-SS microtiter syncytial assay was performed to determine the effects of SF on the anti-HIV activity of MIV-150 and Carraguard.

Back to Top | Article Outline

Statistical Analysis

In all experiments, each concentration of Carraguard, MIV-150, and PC-815 was tested in triplicate. The experiments were repeated at least twice. Standard deviations were calculated and plotted using Microsoft Excel software. Analysis of combined effects was done using the median effect principle. The combination index (CI) was calculated according to the method described by Chou and Talalay.27 The CI values are estimated using the Calcusyn for windows software package (Biosoft, Cambridge, U.K.): additive effect (CI = 1), synergism (CI <1), or antagonism (CI >1) for a given end point of the effect measurement.

Back to Top | Article Outline


Activity of MIV-150 Against HIV-1MN

To demonstrate that our laboratory obtained consistent results for the active concentration with those obtained by Medivir, the activity of MIV-150 to protect target cells from infection was assayed using a syncytial assay. The EC50 of MIV-150 against HIV-1MN in CEM.SS cells was below 0.001 μmol/L (Fig. 1). These data are in agreement with the findings of Medivir (Disa Bottinger, personal communication).

Back to Top | Article Outline

Activity of MIV-150 Against Free Virus

NNRTIs generally act after virus enters susceptible target cells where the RT is released in the cell as opposed to free virus where the RT enzyme is protected by the p24 capsid. To evaluate if MIV-150 inactivated free virus, the syncytial assay was used. MIV-150 effectively inactivated free virus with an EC50 of 0.004 μg/mL (or 0.01 μmol/L) (Fig. 2).

Back to Top | Article Outline

Additive Effect of PC-815

To determine whether there was an additive effect when MIV-150 was combined with Carraguard, various concentrations of each component were tested by detecting the β-galactoside activity in infected cells. The analysis of combined effects using the median effect principle showed values of CI around 1, supporting the idea of an additive effect (Fig. 3; Table 1).

Back to Top | Article Outline

Activity Against Clinical Isolates of HIV

The antiviral activity of Carraguard, PC-815, and methyl cellulose against clade C clinical isolates were compared. At the maximum concentration assayed, 160 μg/mL, methylcellulose had no effect. Carraguard had an EC50 of 77 μg/ml (range, 29–153 μg/mL) for 9 of the 10 clade C viruses tested. There was no effect on the 93/MW/959 virus strain at concentrations below 160 μg/mL. PC-815 blocked all 10 viruses with an EC50 of 7 μg/mL (range, 0.1–14 μg/mL). The average EC50 for PC-815 was approximately 10 times stronger than Carraguard (Table 2).

Back to Top | Article Outline

Activity Against HIV-2

Because HIV-2 infections are prevalent in some regions of West Africa, Carraguard, MIV-150, and PC-815 were assayed for antiviral activity against HIV-2 CDC310342 by quantifying the RT activity in the supernatant of infected cells. The EC50 of Carraguard was 10.5 μg/mL, EC50 of MIV-150 was 0.0003 μg/mL (or 0.001 μmol/L), and EC50 of PC-815 was 1.5 μg/mL (Fig. 4).

Back to Top | Article Outline

Effect of Seminal Fluid on Anti-HIV Activity

Because sexual transmission occurs in the presence of semen, MIV-150 and Carraguard were assayed in the presence of human SF. MIV-150 and Carraguard were assayed separately; the concentration of MIV-150 in PC-815 is very low and would be negligible when the formulation is diluted to offset the toxic effects of SF to cells in the syncytial assay. Toxicity and anti-HIV activity of SF had been determined in a previous experiment and found to be present when the culture medium contains more than 15% or 2% of SF, respectively (data not shown). SF had no effect on the antiviral activity of MIV-150 or Carraguard (Fig. 5A, B).

Back to Top | Article Outline


Although the mechanism(s) of the sexual transmission of HIV have yet to be fully understood, several theories suggest HIV can infect by either cell-associated or free virus.28–31 Given the evidence supporting several mechanisms of transmission, a microbicide capable of blocking infection by targeting different mechanisms of action will have a greater chance of being efficacious against viruses from a broader range of clades. It is also conceivable that a combination formulation may be active against other sexually transmitted pathogens.

MIV-150 was an attractive choice because of the extensive and encouraging data previously obtained with the compound. Scientists have demonstrated MIV-150 is highly effective against HIV and that the drug blocks viruses that are resistant to other marketed HIV antiretrovirals such as nevirapine, delavirdine, and efavirenz. In addition, resistance to MIV-150 develops more slowly than to other anti-HIV drugs tested (Medivir, Disa Bottinger, personal communication). For example, in vitro studies demonstrate that resistance development in the presence of nevirapine and delavirdine takes only 4 to 5 weeks compared with 45 weeks in the presence of MIV-150.

Additionally, in a recently held technical consultation jointly sponsored by the World Health Organization, International Partnership for Microbicides, and the Center for Disease Control and Prevention, it was recommended that the criteria for choosing candidate antiretrovirals for use in a microbicide include little to no cellular toxicity, low oral bioavailability, tight binding, high threshold to resistance development, activity against RT-resistant strains, antiviral activity at low concentrations, stable pH when exposed to a range of temperatures, and a short half-life. These properties are all characteristics of MIV-150.

Extensive animal and human toxicology studies have been carried out, all of which demonstrated that MIV-150 is safe, stable, and well tolerated. Also, it was demonstrated that the drug shows poor systemic absorption, an ideal characteristic for use in a microbicide. MIV-150 is inexpensive to manufacture, further increasing its appeal as a microbicide ingredient.

Data presented here show that Carraguard is effective against 9 of 10 clade C clinical isolates tested at a concentration of less than 160 μg/mL. However, when combined with MIV-150, PC-815 is effective against all clade C clinical isolates and is effective at concentrations an order of magnitude less than Carraguard alone. The actual concentration of carrageenan in undiluted Carraguard is 30,000 μg/mL (approximately 200 times more than the highest concentration used in the PBMC assay). Thus, Carraguard alone displayed strong antiviral activity. Even so, based on these findings, PC-815 should be more effective than Carraguard in preventing infection by clade C viruses.

Microbicide efficacy against HIV-2 is important because although infections by this virus are mainly restricted to West Africa, the prevalence is growing in some areas of Africa, Europe, and Southwest India.32 Some NNRTIs that are effective against HIV-1 are not effective against HIV-2.33 HIV-1 and HIV-2 differ in virus evolution, tropism, and pathogenesis. HIV-2 demonstrates lower pathogenicity compared with HIV-1.32 Related to this lower pathogenicity is an enhanced immune control of HIV-2 infection. Furthermore, CD4-independent infection of target cells can occur with HIV-2, whereas CD4 receptors are integral to infection of main targets (lymphocyte T CD4+ cells and macrophages) for HIV-1.32

All of the formulations tested demonstrated in vitro antiviral activity against HIV-2CDC310342. The in vitro activity of PC-815 against HIV-2 suggests the possibility that the formulation could be evaluated in vivo for activity against SIV or SHIV using the macaque model because HIV-2 is more similar to SIV/SHIV phylogenetically than HIV-1.34 This would be a valuable avenue for future research.

Theoretically, MIV-150 could prevent sexual transmission of HIV in 3 ways. The first would be to enter target cells in the submucosa, bind to and inactive the RT enzyme after the viral capsid has been uncoated. In free virus, the enzyme is associated with the viral genome and is protected by the viral capsid and envelope. However, using the CEM-syncytial assay MIV-150 inactivated free HIV-1MN. This suggests a second and possibly third way by which MIV-150 could prevent HIV infection by either entering a virion and inactivating the RT or by associating with the virion and being carried into a target cell with the virion and subsequently inactivating the RT. Thereby, it could be effective against virus in the vagina before viral infection of the target cells. This is especially important in the case of sexual transmission of HIV because considerable evidence suggests that free virus is captured by dendritic cells and transferred to T-cells locally and/or transported to the lymph nodes.35

Sexual transmission occurs in the presence of SF. It is, therefore, important to determine whether SF will inactivate the active ingredients in the microbicide. In testing possible interference of SF by testing anti-HIV activity of MIV-150 or carrageenan in the presence or absence (control) of human SF, we found that SF had no effect.

In summary, the effectiveness of Carraguard has been improved by adding the NNRTI, MIV-150. The combination product has greater strength against clade C clinical isolates and HIV-2, acts on free virus, and is not inactivated by SF.

Back to Top | Article Outline


1. The Joint United Nations Programme on HIV/AIDS. Microbicides for HIV Prevention: UNAIDS Technical Update. UNAIDS April 1–8, 1998.
2. Pearce-Pratt R, Phillips DM. Sulfated polysaccharides inhibit lymphocyte-to-epithelial transmission of human immunodeficiency virus-1. Biol Reprod 1996; 54:173–182.
3. Perotti ME, Pirovano A, Phillips DM. Carrageenan formulation prevents macrophage trafficking from vagina: Implications for microbicide development. Biol Reprod 2003; 69:933–939.
4. Jerse AE. Experimental gonococcal genital tract infection and opacity protein expression in estradiol-treated mice. Infect Immun 1999; 67:5699–5708.
5. US Food and Drug Administration GRAS (Generally Recognized as Safe) Food Ingredients: Carrageenan. FDA Publications PB-221 206, 1972.
6. Cohen SM, Ito N. A critical review of the toxicological effects of carrageenan and processed eucheuma seaweed on the gastrointestinal tract. Crit Rev Toxicol 2002; 32:413–444.
7. Joint United Nations Programme on HIV/AIDS. Report on the Global HIV/AIDS Epidemic. Geneva, Switzerland: UNAIDS, 2002.
8. International Food Additives Council and Marinalg International/ CLITAM. Carrageenan Monograph. 1991.
9. International Food Additives Council and Marinalg International/ CLITAM. Carrageenan Monograph. 1997.
10. Coggins C, Blanchard K, Alvarez F, et al. Preliminary safety and acceptability of a carrageenan gel for possible use as a vaginal microbicide. Sex Transm Infect 2000; 76:480–483.
11. Phillips DM. Intravaginal formulations to prevent HIV infection. In: Fantini J, Sabatier JM, eds. Perspectives in Drug Discovery and Design, 1996:213–223.
12. Tan X, Phillips DM. Cell-mediated infection of cervix derived epithelial cells with primary isolates of human immunodeficiency virus. Arch Virol 1996; 141:1177–1189.
13. Gendler S, Taylor-Papadimitriou J, Duhig T, et al. A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J Biol Chem 1988; 263:12820–12823.
14. Lynch G, Low L, Li S, et al. Sulfated polyanions prevent HIV infection of lymphocytes by disruption of the CD4-gp120 interaction, but do not inhibit monocyte infection. J Leukoc Biol 1994; 56:266–272.
15. Chakrabarti L, Guyader M, Alizon M, et al. Sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses. Nature 1987; 328:543–547.
16. Jagodzinski PP, Wiaderkiewicz R, Kurzawski G, et al. Mechanism of the inhibitory effect of curdlan sulfate on HIV-1 infection in vitro. Virology 1994; 202:735–745.
17. Nabatov AA, Pollakis G, Linnemann T, et al. Intrapatient alterations in the human immunodeficiency virus type 1 gp120 V1V2 and V3 regions differentially modulate coreceptor usage, virus inhibition by CC/CXC chemokines, soluble CD4, and the b12 and 2G12 monoclonal antibodies. J Virol 2004; 78:524–530.
18. Bartolini B, Di CA, Cavallaro RA, et al. Susceptibility to highly sulphated glycosaminoglycans of human immunodeficiency virus type 1 replication in peripheral blood lymphocytes and monocyte-derived macrophages cell cultures. Antiviral Res 2003; 58:139–147.
19. Zhu T, Mo H, Wang N, et al. Genotypic and phenotypic characterization of HIV-1 patients with primary infection. Science 1993; 261:1179–1181.
20. Essex M. Human immunodeficiency viruses in the developing world. Adv Virus Res 1999; 53:71–88.
21. Nara PL, Hatch WC, Dunlop NM, et al. Simple, rapid, quantitative, syncytium-forming microassay for the detection of human immunodeficiency virus neutralizing antibody. AIDS Res Hum Retroviruses 1987; 3:283–302.
22. Motakis D, Parniak MA. A tight-binding mode of inhibition is essential for anti-human immunodeficiency virus type 1 virucidal activity of nonnucleoside reverse transcriptase inhibitors. Antimicrob Agents Chemother 2002; 46:1851–1856.
23. Jost LM, Kirkwood JM, Whiteside TL. Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells. J Immunol Methods 1992; 147:153–165.
24. Derdeyn CA, Decker JM, Sfakianos JN, et al. Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120. J Virol 2000; 74:8358–8367.
25. Buckheit RW Jr, Watson K, Fliakas-Boltz V, et al. SJ-3366, a unique and highly potent nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1 (HIV-1) that also inhibits HIV-2. Antimicrob Agents Chemother 2001; 45:393–400.
26. Mittman RJ, Bernstein DI, Adler TR, et al. Selective desensitization to seminal plasma protein fractions after immunotherapy for postcoital anaphylaxis. J Allergy Clin Immunol 1990; 86:954–960.
27. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22:27–55.
28. Carreno MP, Chomont N, Kazatchkine MD, et al. Binding of LFA-1 (CD11a) to intercellular adhesion molecule 3 (ICAM-3; CD50) and ICAM-2 (CD102) triggers transmigration of human immunodeficiency virus type 1-infected monocytes through mucosal epithelial cells. J Virol 2002; 76:32–40.
29. Zacharopoulos VR, Perotti ME, Phillips DM. A role for cell migration in the sexual transmission of HIV-1? Curr Biol 1997; 7:534–537.
30. Ibata B, Parr EL, King NJ, et al. Migration of foreign lymphocytes from the mouse vagina into the cervicovaginal mucosa and to the iliac lymph nodes. Biol Reprod 1997; 56:537–543.
31. Khanna KV, Whaley KJ, Zeitlin L, et al. Vaginal transmission of cell-associated HIV-1 in the mouse is blocked by a topical, membrane-modifying agent. J Clin Invest 2002; 109:205–211.
32. Reeves JD, Doms RW. Human immunodeficiency virus type 2. J Gen Virol 2002; 83:1253–1265.
33. Witvrouw M, Pannecouque C, Switzer WM, et al. Susceptibility of HIV-2, SIV and SHIV to various anti-HIV-1 compounds: Implications for treatment and postexposure prophylaxis. Antivir Ther 2004; 9:57–65.
34. Hirsch VM, Olmsted RA, Murphey-Corb M, et al. An African primate lentivirus (SIVsm) closely related to HIV-2. Nature 1989; 339:389–392.
35. Pope M, Haase AT. Transmission, acute HIV-1 infection and the quest for strategies to prevent infection. Nat Med 2003; 9:847–852.
© Copyright 2007 American Sexually Transmitted Diseases Association